The present invention relates to selective and potent. inhibition of the enzyme glutaminase. Compounds are disclosed which efficiently inhibit glutaminase but which have no effect, at higher levels, on various mechanistically and functionally related enzymes. The compounds are useful for neuroprotection and in treatment of hepatic encephalopathy.
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Glutaminase is currently recognized as the most significant glutamine utilizing enzyme present in mammalian central nervous tissue (e.g., the brain and spinal cord). The enzyme catalyzes the conversion of glutamine and water to glutamate, with the production of ammonia.
Following ischemic insult or other traumatic injury to neuronal cells, a number of biochemical changes occur in neuronal tissue surrounding the injured region, including a rise in the extracellular concentration of the excitatory neurotransmitter glutamate. This high concentration of glutamate is believed to be an important factor in delayed neuronal death, in which the ischemic lesion increases approximately 2-fold over a period of time 2-72 hours following the initial ischemic insult. It is believed that this elevated glutamate level exacerbates the primary insult, possibly by acting at excitatory glutamate receptors, and that at least some of the excess glutamate results from enzymatic conversion of glutamine to glutamate by glutaminase.
The maximal activity of glutaminase in the brain is 5-10 xcexcmol/min/g (Alameida et al., 1989). This activity corresponds to a capacity for generating glutamate at a concentration of 5-10 mM each minute, a rate far in excess of the 5-10 xcexcM IC50 for toxicity of glutamate on isolated neurons (Dugan et al., 1995). Therefore, it is apparent that only a very minor fraction of the glutaminase present in the brain needs to be active in a pathological circumstance in order to cause damage. Accordingly, inhibition of glutaminase has been reported as a neuroprotective treatment following ischemic injury (see e.g. Newcomb, PCT Pubn. No. WO 99/09825).
Inhibition of glutaminase may also be used for treatment of hepatic encephalopathy. This condition can arise from chronic liver damage, such as chronic forms of viral hepatitis. Portal blood from the intestine is shunted around the damaged liver, entering the circulation directly. The resulting exposure of the brain to elevated concentrations of blood ammonia produces neurologic symptoms ranging from intellectual impairment and psychiatric symptoms to coma. Several million people are affected to some degree (Hawkins et al.). Current treatments are based on lowering blood ammonia, either by decreasing ammonia generation in the gut, e.g. by treatment with the antibiotic neomycin (Conjeevaram et al.), or by biochemical manipulation of ammonia excretion. For example, the efficacy of ammonia fixation by the urea cycle can be increased by increasing the concentration of ornithine, which is accomplished by inhibiting ornithine amino transferase with a compound such as 5-fluoromethylornithine (Seiler et al.). The use of L-ornithine-L-aspartate has also been reported (Stauch et al.). These treatments, while useful, suffer from toxicity (neomycin) or only partial effectiveness (ornithine compounds).
The role of glutaminase in regulation of blood ammonia is described in Cooper et al. and Oppong et al., and includes the hydrolysis of glutamine to glutamate and ammonia by glutaminase in the intestine. Thus, hepatic encephalopathy could be treated by selective inhibition of glutaminase in intestinal tissue.
To date, no glutaminase inhibitors have been reported that are both potent and specific for glutaminase. Known inhibitors, such as 6-diazo-5-oxo-L-norleucine (xe2x80x9cDONxe2x80x9d; Shapiro et al. 1979) and L-2-amino-4-oxo-5-chloropentanoic acid (xe2x80x9cchloroketonexe2x80x9d; Shapiro et al. 1978; see also Rosenberg, U.S. Pat. No. 5,156,976), also inhibit a variety of glutamine utilizing enzymes, such as amidotransferases (Ahluwahia et aL, 1990).
The present invention includes, in one aspect, a method of selectively inhibiting glutaminase in a cell or tissue, comprising administering to the cell or tissue an effective amount of a compound of formula I: 
where
X is sulfur or oxygen, and R1 and R2 are independently selected from the group consisting of lower alkyl, lower alkoxy, aryl, and xe2x80x94(CH2)n-aryl, where n is 0 or 1, and xe2x80x9carylxe2x80x9d is a monocyclic aromatic or heteroaromatic group, having ring atoms selected from the group consisting of carbon, nitrogen, oxygen, and sulfur, and having at most three non-carbon ring atoms, which group may be unsubstituted or substituted with one or more substituents selected from halogen, lower alkyl, lower alkoxy, amino, lower alkyl amino, amino(lower alkyl), or halo(lower alkyl). R1 and R2 may be the same or different; for ease of preparation, R1 and R2 are the same.
Preferably, X is sulfur. In one embodiment, each of R1 and R2 is xe2x80x94(CH2)n-aryl as defined above. The aryl group may be carbocyclic, e.g. where each of R1 and R2 is phenyl or benzyl, where the ring is unsubstituted or substituted as recited above. Preferred compounds include those in which the ring is unsubstituted or substituted with lower alkyl or lower alkoxy. This group includes exemplary compounds in which each of R and R is benzyl, each of R1 and R2 is p-methoxy phenyl, or each of R1 and R2 is m-tolyl. Compounds with heterocyclic aryl groups include those in which each of R1 and R2 is 2-thiophenyl or each of R1 and R2 is 2-furanyl. Compounds in which R1 and R2 are non-aryl include those in which each of R1 and R2 is lower alkoxy, such as ethoxy, or where each of R1 and R2 is lower alkyl, e.g. t-butyl.
In one embodiment, the tissue is neuronal tissue which has been subjected to ischemia, physical trauma, or a neurodegenerative or neuropsychiatric disorder. Alternatively, the tissue is intestinal tissue, particularly in a subject suffering from hepatic encephalopathy.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.